In mitosis integrins reduce adhesion to extracellular matrix and strengthen adhesion to adjacent cells

To enter mitosis, most adherent animal cells reduce adhesion, which is followed by cell rounding. How mitotic cells regulate adhesion to neighboring cells and extracellular matrix (ECM) proteins is poorly understood. Here we report that, similar to interphase, mitotic cells can employ integrins to initiate adhesion to the ECM in a kindlin- and talin-dependent manner. However, unlike interphase cells, we find that mitotic cells cannot engage newly bound integrins to actomyosin via talin or vinculin to reinforce adhesion. We show that the missing actin connection of newly bound integrins leads to transient ECM-binding and prevents cell spreading during mitosis. Furthermore, β1 integrins strengthen the adhesion of mitotic cells to adjacent cells, which is supported by vinculin, kindlin, and talin1. We conclude that this dual role of integrins in mitosis weakens the cell-ECM adhesion and strengthens the cell-cell adhesion to prevent delamination of the rounding and dividing cell.


Supplementary Fig. 2 | Interphase and mitotic cells initiate and strengthen integrin-mediated
adhesion to ECM proteins. a, Linear fits (lines) with 95% confidence intervals (grey) of adhesion forces to determine the adhesion strengthening rate (AS) of interphase or mitotic STC HeLa cells to Matrigel or BSA ( Fig. 1a and Supplementary Fig. 2b; n ≥ 15 cells per condition and contact time). Dots represent mean adhesion force (±SEM). P-values on fits test slope deviations from 0 and on bars compare whether slopes are different (two-tailed extra sum of squares F-test). b, Adhesion forces of interphase (left) or mitotic STC (right) to BSA and to Matrigel in grey as reference (Fig. 1a). c, Adhesion forces of interphase HeLa cells incubated with β1 integrin blocking-antibodies (clone AIIB2) to Matrigel and of unperturbed HeLa cells (Fig. 1a) in grey as reference. d, Adhesion forces of mitotic STC HeLa cells to Matrigel or BSA and of untreated mitotic HeLa cells to Matrigel in grey as reference (Fig. 1a). e, Adhesion forces of interphase HeLa cells in the presence of 2 µM STC to Matrigel or BSA and of untreated interphase HeLa cells (Fig. 1a) in grey as reference. f-h, Adhesion forces of interphase (left) or mitotic STC (right) MDCK cells (f), fibroblasts (g) or MCF7 cells (h) to given substrates and of respective interphase cells as reference in grey. i, Adhesion forces of HeLa cells in the presence of AIIB2 to Matrigel and untreated mitotic STC HeLa cells (Fig. 1a) in grey as reference. j, Adhesion forces of mitotic STC fibroblast cells to fibronectin fragment FNIII7-10∆RGD and to FNIII7-10 as reference in grey. b-j, Dots represent adhesion forces of single cells, red bars medians and (n) the number of tested cells. AS were determined like in a with the P-value comparing the AS-value to the reference data set. 'Mitotic STC ' indicates that mitotic cells were enriched by STC (Methods). Given P-values calculated using a two-tailed Mann-Whitney test compare displayed adhesion forces with reference data and comparing AS-values to given reference data were calculated by two-tailed extra sum of squares F-tests.

Supplementary Fig. 3 | Verification of adhesome depletion and characterization of integrin and cadherin expression of engineered HeLa cell lines.
Flow cytometry analysis of HeLa (Kyoto), talin1/2-depleted (TKO), kindlin1/2-depleted (KKO HeLa) HeLa (Kyoto) and vinculin-depleted (VKO) HeLa MYH9-GFP H2B-mCherry cells for expression levels of given integrin subunits as well as E-, Nand VE-cadherin. Normalized histograms of fluorescence intensities for interphase or mitotic STC HeLa cells stained with antibodies against the given integrin subunit or cadherin are shown. Negative controls were unstained cells. 50'000 cells were analyzed for each condition.

Supplementary Fig. 4 | Increased cell-cell adhesion of mitotic cells is maintained across cell
lines. a, Adhesion strengthening over time is determined as a linear regression of adhesion forces for all contact times for given conditions ( Fig. 1g; n ≥ 12 cells per condition and contact time). Dots represent means (±SEM) of adhesion forces. Lines depict fit and grey areas their 95% confidence intervals. P-values on fits test slope deviations from 0 and on bars compare whether slopes are different (two-tailed extra sum of squares F-test). b, Cell-cell adhesion forces between two interphase HeLa cells in the presence of 2 µM STC (Fig. 1g). Dots represent adhesion forces of single cells, red bars median values and (n) the number of tested cells per condition. As reference adhesion forces between untreated interphase HeLa cells are given (Fig. 1g). AS-values give the adhesion strengthening rate as the slope (±SE) of a linear fit through adhesion forces for all contact times with the P-value comparing the AS-value to that of the reference data set. c, Adhesion forces between an untreated mitotic and an interphase HeLa cell. Data representation as in b. As reference, cell-cell adhesion forces between an interphase and mitotic STC HeLa cells are given in grey (Fig. 1g). d, Cell-cell adhesion forces between two interphase (left), an interphase and a mitotic STC (middle) and two mitotic STC (right) MDCK cells. Data representation as in b. Cell-cell adhesion forces between two interphase MDCK cells are given as reference in grey for comparison in the middle and right panel. e, Adhesion forces between two interphase (left) or an interphase and a mitotic STC (right) MCF7 cell. Data representation as in b. Adhesion forces between two interphase MCF7 cells are given as reference in grey. b-e, Mitotic STC cells were enriched by 2 µM STC for 12 h prior to and incubated with STC throughout the experiments. Pvalues comparing adhesion forces were calculated using two-tailed Mann-Whitney tests and compare displayed adhesion forces with given reference condition. P-values comparing AS-values were calculated by a two-tailed extra sum of squares F-test and compare AS-values of given and reference data.

Supplementary Fig. 5 | Verification of adhesome protein depletion in HeLa cell lines. Cell lysates of control HeLa cells and HeLa cells depleted from vinculin (VKO), talin1/2 (TKO) or kindlin1/2 (KKO)
were immunoblotted (n = 1). Wildtype HeLa cells were used as positive controls, GAPDH was used as loading control. Fig. 1a, Fig. 3, Fig. 5 and Fig. 6; n ≥ 12 cells per condition and contact time). For each condition a linear regression of adhesion forces for all contact times determined the adhesion strengthening over time. Dots represent means (±SEM) of adhesion forces at given contact time. Lines depict the fit and the grey area the 95% confidence interval of the fit. P-values on fits test whether slopes deviate from 0. P-values on bars compare whether slopes of two fits are significantly different (two-tailed extra sum of squares F-test).

Supplementary Fig. 7 | Re-expression of specific adaptor proteins in KO HeLa cell lines recovers adhesion defects. a-c,
Cell-ECM adhesion forces to Matrigel or BSA of (left) interphase and (right) mitotic STC knock-out HeLa cells re-expressing the depleted protein adhesome vinculin (VKO+vinculin) (a), talin1 (TKO+talin1) (b) or kindlin2 (KKO+kindlin2) (c) after given contact times. Dots represent adhesion forces of single cells, red bars median values and (n) the number of tested cells per condition. As reference, cell-ECM adhesion forces of control HeLa cells are given in grey (Fig. 1a). AS-values give the adhesion strengthening rate as the slope (±SE) of a linear fit through adhesion forces for all contact times with the P-value comparing the AS-value to that of the reference data set. P-values compare the displayed data with the reference data given. d-f, Cell-cell adhesion forces between VKO+vinculin (d), TKO+talin1 (e) or KKO+kindlin2 (f) HeLa cells and control HeLa cells spread on a Matrigel coated substrate after given contact times. Panels show adhesion forces of respective interphase re-expressing and interphase control (left), mitotic STC re-expressing and interphase control (middle) or mitotic STC re-expressing and mitotic STC control (right) HeLa cells. As reference cell-cell adhesion forces established between two control HeLa cells for the respective condition is given in grey (Fig. 1g). Data representation as in a. P-values compare displayed data with given reference data. Mitotic cells were enriched by 2 µM STC for 12 h prior to and incubated with STC throughout the experiments. P-values comparing adhesion forces with given reference condition were calculated using two-tailed Mann-Whitney tests and comparing AS-values to given reference data were calculated by two-tailed extra sum of squares F-tests.

Supplementary Fig. 8 | Kindlin and talin are essential for mitotic cell-ECM adhesion in fibroblasts.
Cell-ECM adhesion forces of interphase (left) or mitotic STC (right) TKO (a), KKO (b) or TKO+THD (c) fibroblasts to FNIII7-10 at given contact times. Adhesion forces of wild type interphase or mitotic STC fibroblasts to FNIII7-10 are given as reference in grey (Supplementary Fig. 2g). Dots represent adhesion forces of single cells, red bars median values and n (cells) number of tested cells per condition. AS-values give the adhesion strengthening rate as the slope (±SE) of a linear fit through adhesion forces for all contact times with the P-value comparing the AS-value to that of the reference data set. If only one row P-values are given they compare given and reference data. Otherwise, top row P-values compare mitotic STC adhesion forces with respective interphase adhesion forces and bottom row P-values compare given and reference adhesion forces. 'Mitotic STC ' indicates that mitotic cells were enriched by 2 µM STC for 12 h prior to and incubated with STC throughout the experiments. P-values were calculated using two-tailed Mann-Whitney tests and P-values comparing AS-values were calculated by a two-tailed extra sum of squares F-test.  Fig. 1d using a two-tailed Mann-Whitney test.